GSM7383453: RNA-seq of WT cells after 30 h growth in MMM supplemented with 25 mM NAG solid medium at 28°C - replicate 1; Streptomyces ambofaciens ATCC 23877; RNA-Seq
Remark : Librairy prepared with Total RNA Stranded (reversely stranded) kit => The R1 file corresponds to antisense orientation, and R2 to the sense orientation. Cells were then harvested through centrifugation for 15 min at 4,000 g at 4°C, and stored at -20°C. Pellets were washed with 1 ml of DEPC water, centrifuged for 5 min at 16,000 g at 4°C, and homogenized with glass beads (≤ 106 µm; G4649, SIGMA) in 350 µl of lysis buffer (RNeasy Plus Mini Kit, QIAGEN) supplemented with 10 µl/ml β-mercaptoethanol. Samples were processed 3 times for 45 sec each in FastPrep-24TM 5G instrument (MP Biomedicals) at setting 6 with 1 min cooling between the stages. After centrifugation for 10 min at 16,000 g at 4°C, total RNAs were isolated from the supernatants using RNeasy Plus Mini Kit (QIAGEN) and gDNA Eliminator columns, following manufacturer's recommendations. To remove genomic DNA, RNA samples were incubated from 30 min at 37°C with 20 U of RNase-free DNase I (Roche) in a final reaction volume of 30 µl. RNA were then cleaned up using the RNeasy Mini Kit (QIAGEN), following manufacturer's recommendations. The absence of DNA in the preparations was checked by PCR on an aliquot. RNA samples were quantified using QubitTM RNA HS Assay kit (ThermoFischer Scientific), following manufacturer's recommendations. Total RNA quality was assessed on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies). 500 ng of total RNA were treated with DNAse (Baseline Zero DNAse, Epicentre) prior to ribosomal RNA depletion using the RiboZero bacteria magnetic Kit from Illumina according to the manufacturer recommendations. After the Ribo-Zero step, the samples were checked on the Agilent Bioanalyzer for proper rRNA depletion. Directional RNA-seq libraries were constructed using the Illumina Stranded library preparation kit, according to the manufacturer recommendations.  Libraries were pooled in equimolar proportions and sequenced (Paired-end 2x43 bp) on an Illumina NextSeq500 instrument, using a NextSeq 500 High Output 75 cycles kit.